Postsynaptic densities (PSDs) organize various elements of the postsynaptic response and are likely to play a role in the modification of synaptic efficacy. Calcium-induced changes in PSDs may be involved in the induction and maintenance of activity-dependent synaptic modification. By combined biomedical morphological methods, the project seeks to determine the molecular architecture of the PSD and to identify possible mechanisms for modification by elevated postsynaptic levels of calcium. A major focus has been on CaM kinase II, the most abundant protein in the PSD preparation and a calcium-regulated enzyme implicated in LTP and memory. Previous studies using isolated PSDs indicated that the PSD-associated CaM kinase pool is fully active and has unique properties. In collaboration with Dr. Jaffe, studies to identify autophosphorylation sites of the enzyme are continuing. In addition to Thr 253, described previously, a CnBr cleavage product containing at least three additional potential phosphorylation sites has been identified. Based on the property of CaM kinase II to be phosphorylated at distinct sites in the presence and absence of calcium, a model for its regulation has been devised. A computer simulation of the model (in collaboration with Dr. Albers, LNC) suggests that the autophosphorylation state of an array of CaM kinase II molecules can reflect the frequency of the calcium signal. An important implication of the model is that it provides a potential mechanism for decoding the temporal pattern of synaptic activity. Present studies using subcellular fractions are aimed at testing various implications of the above model. Organotypic hippocampal cultures, developed by Dr. Miyaguchi, are now available to study the regulation of CaM kinase II in intact tissue. Since ischemic has been reported to cause translocation of cytosolic CaM kinase II to the PSDs, we are investigating biomedical and morphological changes associated with ischemic insult to hippocampal cultures. Preliminary results indicate a selective proteolysis following 10 min under ischemic conditions. Future PSD-associated CaM kinase II following specific treatments and to correlate the autophosphorylation state with structural modification.